PROJECT SUMMARY The opioid epidemic claims more than 50,000 lives every year and contributes to a significant drop in overall life expectancy in the USA. The primary cause of death associated with opioid-based analgesics and drugs of abuse is Opioid-mediated Respiratory Suppression (ORS). Although, the mortality risk increases in a dose- dependent manner, opioid use is particularly dangerous because it is unpredictable. Many conditions increase the vulnerability to opioids, including sleep disordered breathing, which is very common among opioid users. Opioids cause respiratory depression and terminal apnea by inhibiting rhythmogenic networks within the ventrolateral medulla. This project has 4 aims to explore the medullary mechanisms underlying ORS. Aim 1 employs a variety of electrophysiological, pharmacological, and optogenetic approaches in vitro and in vivo to explore how opioids inhibit the inspiratory rhythmogenic network. This opioid-sensitive network forms a column that dynamically extends beyond the well-known preBtzinger complex, a microcircuit that is essential for breathing. Aim 2 will obtain horizontal slices from this rhythmogenic column to dissect the pre-and postsynaptic mechanisms that are responsible for the cessation of inspiratory activity. Aim 3 will investigate how opioids inhibit Postinspiration within the postinspiratory complex (PiCo), an excitatory network that is an order of magnitude more sensitive to opioids than the preBtC. The mechanisms revealed in aim 1-3 will provide the basis for aim 4, which will explore combinations of substances capable of reversing ORS in alert animals. This project introduces novel concepts of respiratory rhythmogenesis and describes multiple mechanisms of opioid actions, which could explain why opioid respiratory depression is so unpredictable. We test how opioid modulation is sensitized by hypercapnic conditions and chronic intermittent hypoxia, both conditions are often experienced by opioid users. The proposed research may lead to a better understanding of the mechanisms underlying the mortality and morbidity associated with the opioid crisis.